The long-term goal of our research is to understand the molecular mechanism of senescence and immortalization in human oral epithelial cells. Cellular senescence is an irreversible arrest of cell divisions that results in normal somatic cells after completing a finite number of cell divisions. Several theories of senescence have been proposed, including DNA damage theory, mitochondrial theory, and telomere hypothesis. These theories of aging focus either on (1) extrinsic environmental factors or (2) intrinsic genetic programming as the cause of cellular senescence. Among the extrinsic factors, oxidative stress imposes the most detrimental effect on cells and subcellular macromolecues due to generation of reactive oxygen species (ROS). In contrast, the telomere hypothesis maintains the important role of telomere shortening in senescence of cells. Both theories of senescence mechanism have been experimentally validated to some extent, although exceptions do exist in some cell systems. To determine the mode of senescence in normal human oral keratinocytes (NHOK), we established an in vitro model of senescence, in which the cells undergo 22+/- 3 cell divisions before senescence. Unlike fibroblasts, replicating NHOK demonstrated telomerase activity and maintained constant length of telomeres, suggesting telomere length-independent mode of senescence in these cells. In our recent study, we also found that senescence of NHOK enhanced expression of mitochondrial proteins and increased mitochondrial mass, which reflect oxidative stress response in cells. Simutaneously, the expression levels of DNA repair genes were down-regulated in senescing NHOK. Therefore, our data point to the linkage between oxidative stress, DNA damage, and senescence in NHOK. The central hypothesis of the current proposal is: Senescence of NHOK is triggered by accumulation of DNA damage that results from accumulation of oxidative stress and impaired DNA repair capacity of cells. To test this hypothesis, we propose to: (1) determine the phenotypic and molecular alterations of NHOK in response to oxidative stress, (2) investigate the changes in the DNA repair capacities of NHOK during in vitro life span with or without oxidative stress, and (3) determine the effect of disrupting the DNA repair pathways on phenotypic and molecular alterations of NHOK in response to oxidative stress. With these Aims, we will address the questions: What is the effect of oxidative stress on replication and senescence of NHOK? Is senescence associated with reduction in DNA repair activities and accumulation of DNA damage in NHOK? What is the role of mitochondria in senescence of these cells? Can we disrupt the DNA repair capacities in NHOK and trigger premature aging in cells? Can the cells with impaired DNA repair activities be rescued from premature senescence by antioxidants?